Effects of prime-boost strategies on the protective efficacy and immunogenicity of a PLGA (85:15)-encapsulated Chlamydia recombinant MOMP nanovaccine

Abstract To begin to optimize the immunization routes for our reported PLGA-rMOMP nanovaccine [PLGA-encapsulated Chlamydia muridarum (Cm) recombinant major outer membrane protein (rMOMP)], we compared two prime-boost immunization strategies [subcutaneous (SC) and intramuscular (IM-p) prime routes followed by two SC-boosts)] to evaluate the nanovaccine-induced protective efficacy and immunogenicity in female BALB/c mice. Our results showed that mice immunized via the SC and IM-p routes were protected against a Cm genital challenge by a reduction in bacterial burden and with fewer bacteria in the SC mice. Protection of mice correlated with rMOMP-specific Th1 (IL-2 and IFN-γ) and not Th2 (IL-4, IL-9, and IL-13) cytokines, and CD4+ memory (CD44highCD62Lhigh) T-cells, especially in the SC mice. We also observed higher levels of IL-1α, IL-6, IL-17, CCL-2, and G-CSF in SC-immunized mice. Notably, an increase of cytokines/chemokines was seen after the challenge in the SC, IM-p, and control mice (rMOMP and PBS), suggesting a Cm stimulation. In parallel, rMOMP-specific Th1 (IgG2a and IgG2b) and Th2 (IgG1) serum, mucosal, serum avidity, and neutralizing antibodies were more elevated in SC than in IM-p mice. Overall, the homologous SC prime-boost immunization of mice induced enhanced cellular and antibody responses with better protection against a genital challenge compared to the heterologous IM-p.


Introduction
De v elopment of a safe and effectiv e v accine is onl y the first step to control a human infectious pathogen.In recent years, vaccine de v elopment efforts a gainst human pathogens ar e r a pidl y pr ogressing to w ar d biodegradable nanoparticle-based delivery of encapsulated subunit antigens (Lung et al. 2020 ).Due to the weak immunogenicity of subunit vaccines, they require a delivery system, an adjuvant to bolster immune responses, and often multiple dosages to induce adequate protective immune responses (Singh et al. 2015, Tsoras and Champion 2019, Kumar et al. 2020 ).Vaccine-induced immunity is also profound upon the immunization route to elicit anamnestic immune responses (Estcourt et al. 2005, Herzog 2014, Pais et al. 2019 ).Targeting mucosal routes for immunization has been effective since most infections occur through mucosal surfaces (Holmgren and Czerkinsky 2005 ).Ne v ertheless, m ultiple studies have indicated that nonmucosal routes can induce better immune responses for vaccines against some cancers (Chen et al. 2018(Chen et al. , 2019 ) ), bacteria (Khan et al. 2017, Khademi et al. 2019 ), viruses (Lin et al. 2014, Gebauer et al. 2019 ), and parasites (Noormehr et al. 2018, Pandey et al. 2018 ).Currently, many subunit vaccines that are in preclinical development interc hangeabl y use systemic and m ucosal r outes or a combination of both for immunization (Holmgren et al. 2013, 2018, Manoff et al. 2015, Kumar et al. 2020, Lakatos et al. 2020 ).
Chlam ydia trac homatis (Ct) is the most common etiologic a gent of bacterial sexually transmitted diseases, leading to considerable r epr oductiv e morbidities worldwide (Low et al. 2016 ).Gener all y, females ar e mor e pr one to long-term persistent infections that pose significant risks, often causing pelvic inflammatory disease, infertility , ectopic pregnancy , and c hr onic abdominal pain (Cluver et al. 2017, Poston et al. 2019 ).Reportedl y, Chlam ydia infections can boost human immunodeficiency virus (HIV) transmission and may serve as a cofactor in human papillomavirus-induced cervical neoplasia (Simonetti et al. 2009, Jensen et al. 2014, Masia et al. 2020 ), thereby posing a considerable burden on public health globally.Despite these significant public health challenges, there is no a ppr ov ed c hlamydial v accine .T he de v elopment of a v accine a gainst genital Chlam ydia could gr eatl y aid in the amelioration of the induced morbidities and comorbidities.
Chlamydia is an infectious pathogen whereby vaccine-induced immunity is exceptionally challenging since an attenuated or inactiv ated whole c hlamydial elementary bodies (EBs) vaccine is not practical due to the induction of immunopathology (Mabey et al. 2014 ).Also, the need for ser ov ar-specific pr otection (de la Maza et al. 2017 ) further stifles the pr ocess.Av ailable e vidence indicates that protection against Chlamydia involves coordination from cellmediated and humoral immunity such as CD4 + T-cells , T h1secreting cytokines (i.e.IFN-γ , IL-2), and antibodies (IgG and IgA), to clear the bacterial infection (Farris et al. 2010, Fiorino et al. 2013, Lorenzen et al. 2015, Wern et al. 2017 ).Other investigators show that an immunomodulatory Th17 response also plays a role in Chlam ydia v accine-induced imm unity (Vicetti Miguel et al. 2016 ).
Chlamydia major outer membrane protein (MOMP) has been studied for years and is a prime subunit v accine tar get because it is immunogenic and elicits cellular and humoral immune responses that are requisites for protective immunity against genital Chlamydia (O'Meara et al. 2013, de la Maza et al. 2017, Poston et al. 2019 ).Recombinant MOMP adjuvanted with DDA/MPL and chlamydial Pmps (Yu et al. 2014 ), CAF01, and CAF09 (Pal et al. 2017 ), TLR agonists (Cheng et al. 2011, 2014, Pal et al. 2020, Tifrea et al. 2020 ) or c holer a toxin subunits (Singh et al. 2006, Ekong et al. 2009 ) have all protected mice against genital Chlamydia .Nonetheless, the protection afforded by the MOMP vaccine candidates is short-term and does not induce sterilizing or long-lasting protectiv e imm unity, pr obabl y because of ineffectiv e adjuv ants to bolster m ucosal imm une r esponses (Singh et al. 2006, Stary et al. 2015 ), efficient delivery systems (Dixit et al. 2018 ), or inadequate r outes of administr ation (Fiorino et al. 2013, Lor enzen et al. 2015, Pais et al. 2019 ).
The v accine-deliv ery r oute has a significant impact on the induction of efficacious host immune responses .Moreo ver, an optimal v accine-deliv ery system can pr ofoundl y dictate the outcome of the elicited immune responses.Efforts to develop and optimize a vaccine against Chlamydia have sought numerous delivery routes (Berry et al. 2004, Ralli-Jain et al. 2010, Pais et al. 2017 ) and prime-boost immunization strategies (Brown et al. 2012, Lorenzen et al. 2015, Badamchi-Zadeh et al. 2016 ).Our effort in the preclinical de v elopment of a Chlam ydia v accine has focused primarily on delivery systems using biodegradable-polymeric nanoparticles with self-adjuvanting properties.Using such an adjuvantdelivery system, we have successfully developed several potential Chlam ydia nanov accine candidates a gainst MOMP or its peptides (Taha et al. 2012, Fairley et al. 2013, Dixit et al. 2014, Verma et al. 2018, Sahu et al. 2020 ).We r ecentl y r eported that our c hlamydial PLGA-rMOMP nanovaccine consisting of recombinant major outer membr ane pr otein (rMOMP) enca psulated in extended-r eleasing PLGA (85:15) nanoparticles-trigger ed activ ation of dendritic cells to pr oduce r obust T h1 cytokines , ada ptiv e imm une r esponses, and MHC-II antigen presentation (Sahu et al. 2020 ).Our data sho w ed that PLGA-rMOMP administered via a homologous prime-boost subcutaneous (SC) route protected mice against a Chlamydia muridarum (Cm) genital challenge but failed to confer complete protection.Given the impact of prime-boost immunization routes on a v accine's pr otectiv e potential, her ein, we determined the impact of the SC homologous versus a heterologous intramuscular (IMp) prime-boost immunization on the PLGA-rMOMP nanovaccineinduced immunogenicity and protective efficacy against a Cm genital c hallenge .Her e, we pr esent and discuss our r esults fr om the prime-boost studies conducted in the female BALB/c mouse model.

PLGA-rMOMP nano v accine formula tion
T he PLGA-rMOMP nano v accine was form ulated, as pr e viousl y r eported (Sahu et al. 2020(Sahu et al. , 2021 ) ).Briefly, PLGA 85:15 (150 mg) was dissolved in DCM, follo w ed b y the addition of 2 mg of rMOMP, homogenization, and then the addition of 1% PVA.The resulting doubleem ulsion was gentl y stirr ed ov ernight at r oom temper atur e (RT) to allow e v a por ation of the DCM organic solvent, harvested by ultracentrifugation, washed, and then lyophilized in the presence of a 5% trehalose solution.Lyophilized nanoparticles were stored at −80 • C in a sealed container until used.

Mice immunization and challenge
Female BALB/c mice (4-6 weeks old) were purchased from Charles Riv er Labor atory (Raleigh, NC, USA) and housed under standard pathogen-fr ee and contr olled envir onmental conditions and provided with food and water ad libitum .Mice were acclimatized for 2-weeks before all experimental procedures as approved by Alabama State University Institutional Animal Care and Use Committee (IACUC).Mice were divided into experimental groups (12 mice/group) for the immunization studies and were primed on day 0 via the IM-p (heterologous) or SC (homologous) routes with PLGA-rMOMP (50 μg).Two boosters of PLGA-rMOMP (50 μg) were administered via the SC route on days 14 and 28 (Fig. 1 A).A total of 2-weeks following the last immunization (day 42), 6 mice/group were sacrificed to collect spleen, serum, and mucosal wash samples for analyses of cellular and humoral immune responses, respectiv el y.Mice in the PBS and rMOMP gr oups, r espectiv el y, wer e administered SC with 100 μl of sterile PBS or 50 μg of rMOMP.
For the challenge studies, immunized mice (six per/group) were eac h administer ed 2.5 mg of Depo-Pr ov er a (day 42) SC and c hallenged (day 49) intr av a ginall y with 1 × 10 5 IFU of Cm in sucrose phosphate glutamate (SPG) buffer (Verma et al. 2018, Sahu et al. 2021 ).Cervico-v a ginal swabs were collected at 3-day intervals for 3-w eeks, and mice w ere sacrificed on da y 70 (Fig. 1 B).All s wabs were collected in SPG buffer and stored at −80 • C to quantify the Cm v a ginal bacterial burden (Verma et al. 2018, Sahu et al. 2021 ).

Quantification of Cm from vaginal sw a bs
Sw abs w er e pr opa gated in McCo y cell monolay ers containing 0.5 μg/ml cycloheximide, centrifuged at 750 × g for 1 h at RT and then incubated for 2 h at 37 • C in a 5% CO 2 humidified atmosphere.After that, the media was replaced with fresh media containing 0.5 μg/ml cycloheximide and further incubated for 30 h.The cells w ere w ashed, fixed in 95% ethanol, and stained with a FITC-labeled Chlamydia antibody using the Remel TM PathoDx TM Chlam ydia Cultur e confirmation kit.Inclusions wer e ca ptur ed using a fluorescent microscope (Nikon, Melville, NY, USA), visually counted and calculated as IFU/ml (Verma et al. 2018, Sahu et al. 2021 ).

Antigen-specific T-cells prolifer a tion, and memory and effector phenotypes quantification
Spleens were pooled per group and k e pt in RPMI-1640 supplemented with 10% FBS and antibiotics-antimycotic. Single-cell suspensions were obtained and filtered through a 40-micron nylon mesh strainer and washed before red blood cells lyses using ACK lysing solution.Total T-cells were purified from splenocytes with anti-CD 90.2-conjugated magnetic beads by positive selection over MACS columns and subjected to CFSE-based proliferation assay, as pr e viousl y described (Dixit et al. 2018, Verma et al. 2018, Sahu et al. 2020, 2021 ).CFSE-labeled T-cells (1 × 10 6 ) were cocultured with mitomycin-C (25 μg/ml) treated APCs (1 × 10 6 ) and stimulated with rMOMP (5 μg/ml) in round bottom-polypropylene tissue culture tubes and incubated for 120 h at 37 • C in a 5% CO 2humidified atmosphere.Cells were harvested and stained using CD3-APC-Cy7, CD4-PerCP-Cy5.5, CD62L-APC, and CD44-PE to e v aluate T-cells pr olifer ation, and memory (CD44 high CD62L high ) and effector (CD44 high CD62L low ) phenotypes.Following the staining, cells w ere w ashed, fixed, and data wer e acquir ed on a BD LSR II flo w c ytometer and analyzed using FCS Express FLOW6 (De Novo Softwar e, P asadena, CA, USA).Gating on CFSE + T-cells was used for the selection of CD3 + CD4 + T-cell populations ( Figures S1 -S8 , Supporting Information ).Histogr am fluor escence intensities wer e used to quantify the pr olifer ating and r esting T-cells amongst the total CFSE + CD3 + CD4 + T-cells.

Cytokines quantification
Purified T-cells were co-cultured with APCs and stimulated with rMOMP (5 μg/ml), and cell-fr ee cultur e supernatants wer e collected at 120 h by centrifugation for cytokines quantification, as described pr e viousl y (Dixit et al. 2018, Verma et al. 2018, Sahu et al. 2020, 2021 ).T he T h1 and T h2 c ytokine ratios w ere calculated using the following equation:

Quantification of antigen-specific serum and mucosal antibody isotypes
Antibody isotypes (IgG2a and IgG2b (Th1) and IgG1 (Th2)) A were quantified from pooled sera or vaginal wash (including IgA) samples, as described pr e viousl y (Singh et al. 2006, Fairley et al. 2013, Dixit et al. 2014, Verma et al. 2018, Sahu et al. 2021 ).Briefly, ELISA plates were coated with 100 μl (1 μg/ml) of purified rMOMP and k e pt o vernight at 4 • C. T he rMOMP-coated plates were then washed with PBS-Tween 20 (PBST) and blocked in 3% nonfat dry milk.In a separate plate, samples were serially diluted (2-fold), starting at 1:4000 (serum IgG1), 1:500 (serum IgG2a and IgG2b), 1:25 (mucosal wash IgG1, IgG2a, and IgG2b), and 1:5 (mucosal wash IgA) to determine the endpoint titers.Antigen-specific IgG2a and IgG2b (Th1) and IgG1 (Th2) antibodies were detected using isotype-specific HRP-conjugated antibodies (goat antimouse) and TMB substrate .T he endpoint titer was considered to be the last sample dilution with readings higher than the mean + 5 standard deviations (SD) of the negative control serum or vaginal wash (IgG isotypes) or the mean + 3 (SD) of the negativ e contr ol v a ginal wash samples (IgA).All samples were run in triplicates, and experiments were repeated at least three times .T he T h1 and Th2 antibody ratios were calculated using the following equation:

Quantification of antibody isotypes avidity index
Serum antibody isotypes avidity index (AI) was determined as previously described (Verma et al. 2018, Sahu et al. 2020, 2021 ).ELISA plates were coated with purified rMOMP, as described above in the serum and mucosal antibodies section.Sera were diluted (1:50, 1:100, 1:200, and 1:400) and then added to wells in parallel (two sets per plate) and incubated for 2 h at RT. Plates were washed with PBST, and one set for each sample was treated with urea (8 M in PBST), and the other set was treated with PBST for 5 min at RT.After washing, rMOMP-specific IgG2a and IgG2b (Th1) and IgG1 (Th2) isotypes AI was detected using isotype-specific HRPconjugated goat antimouse antibodies and TMB substrate .T he experiments wer e r epeated at least two times, and each sample was run in triplicates .T he AI was calculated using the following equation:

Neutr aliza tion of Chlamydia in vitro
Neutralization of Cm EBs by ser a fr om imm unized (pr e) and imm unized-c hallenged (post) mice was performed in McCoy cells, as pr e viousl y described (Verma et al. 2018, Sahu et al. 2021 ).Briefly, McCoy cell monolayers were infected with EBs (pretreated with sera) by centrifugation for 1 h at 750 × g and incubated for 30 h in a 37 • C incubator.Cells were fixed, stained with Chlamydia confirmation kit (Remel, ThermoFisher, USA).Inclusions wer e ca ptur ed using a fluorescent microscope (Nikon, Melville, NY, USA), counted and calculated as IFU/ml.

Sta tistical anal ysis
Data were analyzed b y tw o-w ay analysis of variance (ANOVA) follo w ed b y Tuk e y's m ultiple comparison to compar e the number of Cm IFU, and cellular and humoral immune responses from rMOMP, SC and IM-p using GraphPad Prism 10 (San Diego, CA, USA) to observe the differences between immunized (pr e), imm unized-c hallenged (post) and control groups .One-wa y ANOVA was used for the av er a ge of total IFU to compare the % reduction in recovered IFU between immunized-challenged (post) gr oups.P -v alues ≤ .05were considered statistically significant.

Homologous (SC) is more effecti v e than heterologous (IM-p) prime-boost immunization against clearance of genital Chlamydia
Exploring various prime-boost immunization strategies in the pr eclinical de v elopment of an efficacious v accine a gainst genital Chlamydia is essential (Brown et al. 2012, Badamchi-Zadeh et al. 2016 ).To begin to optimize the immunization routes for our c hlamydial PLGA-rMOMP nanov accine, we used two prime-boost imm unization str ategies to compar e the nanov accine-induced pr otectiv e efficacy and immunogenicity in mice.As depicted in Fig. 1 , mice r eceiv ed PLGA-rMOMP either via the IM-p (heter ologous) or SC (homologous) routes follo w ed b y tw o SC route booster immunizations and then a challenge via the mucosal intravaginal route with Cm IFU (1 × 10 5 ).Cervico-v a ginal sw abs w ere collected at 3-day intervals up to 3-weeks to evaluate protection by quan-

Nano v accine-induced antigen-specific cellular immune responses in mice
It is w ell-kno wn that cell-mediated immunity, as elicited by a vaccine, is k e y to pr otecting a gainst genital Chlam ydia with activ ated T-cells and Th1 cytokines serving as pivotal pr ota gonists (Bakshi et al. 2018, Helble et al. 2020 ).We e v aluated T-cell-mediated immune effectors that may correlate with PLGA-rMOMP protectiv e efficacy a gainst genital Chlam ydia in imm unized mice.Purified splenic T-cells from immunized (pre) and immunized-challenged  (post) mice were co-cultured with mitomycin-C treated APCs and stimulated with rMOMP for 120 h.Post-stimulation, cell-free supernatants were collected and used to quantify various cytokines/c hemokines that ar e necessary for clear ance of c hlamydial burden in the genital tract (Helble et al. 2020 ).
As depicted in Fig. 3 (A), T-cells from SC-and IM-p-immunized mice (pre) were found to significantly ( P < .0001)produce more Th1 cytokines (IL-2, and IFN-γ ) compared to those from the rMOMP and other control groups of mice.It is worth noting that both cytokines were significantly enhanced ( P < .0001)after a Cm genital challenge (post).
We also observed (Fig. 3 B) that T-cells from the SC and IMp immunized mice (pre) produced significantly ( P < .001)higher le v els of rMOMP-specific IL-6 (a pro-inflammatory cytokine) compared to the control groups (pre).After a genital challenge (post), T-cells from all groups secreted high levels of IL-6, with SC mice showing the highest le v el.IL-17 pr oduction was significantl y high ( P < .0001) in the rMOMP-, SC-, and IM-p-imm unized (pr e) gr oups.Lo w er le v els of IL-17 was observ ed in the negativ e contr ol gr oups.We also observed enhanced IL-17 production ( > 8-fold) in all groups of mice after the Cm genital challenge (post).
Figure 3 (C) shows that CCL-2 (also known as monocyte c hemoattr actant pr otein 1 (MCP1)) pr oduction was significantl y ( P < .05)induced in SC-and IM-p-immunized mice (pre), which decreased after Cm genital challenge (post).The granulocyte colonystimulating factor (G-CSF) (Fig. 3 C), a stimulator of stem cells to pr oduce mor e leuk oc ytes, w as significantly ( P < .0001)high in SC-and IM-p-immunized mice (pre).In addition, we noticed that the SC mice produced 2-fold higher levels of G-CSF in comparison to the IM-p mice .T he production of G-CSF was significantly ( P < .0001)enhanced after a Cm genital challenge (post) but interestingl y, slightl y higher in IM-p mice compared to SC mice (post).
In Fig. 3 (D), it can be seen that T-cells from SC-and IM-pimmunized mice (pre) secreted significantly ( P < .0001)higher IL-1 α than the rMOMP and other control groups.After challenge (post), the le v els of IL-1 α wer e significantl y ( P < .0001)enhanced for SC, follo w ed b y IM-p and rMOMP compared to the negative contr ol gr oups .T he pr oduction of IL-4, a pr ototype T h2 cytokine , w as lo w in SC-, IM-p-, and rMOMP-imm unized mice (pr e), and was further reduced in challenge mice (post).On the other hand, IL-13 pr oduction was ele v ated significantl y ( P < .001) in SC (post) compared to all other groups (pre and post).Additionally, IL-9 production was significantly ( P < .0001)induced in all pre and post gr oups, especiall y in SC.Ov er all, Th2 cytokines were lower than Th1 cytokines in SC and IM-p mice.The PLGA-PBS negative contr ol r esponses wer e similar to those of PBS; thus, thus statistical comparison was not included in the gr a phs for clarity.
Assessment of a chlamydial vaccine efficacy also entails evaluating the v accine's ca pacity to induce activation of T-cells and the formation of memory and effector cells .Here , the ability of PLGA-rMOMP to activate T-cells after immunization and challenge of mice was investigated.We focused on CD4 + T-cells proliferation and differentiation into memory (CD44 high CD62L high ), and effector (CD44 high CD62L low ) phenotypes that contribute to bacterial clear ance.Our compar ativ e r esults, as depicted in Fig. 5 (A)-(L), show that after imm unization (pr e), T-cell activ ation was in the order of magnitude SC > IM-p > rMOMP > PBS.The increase in the CD4 + T-cell percentages was comparable for SC (46.90%) and IM-p (45.27%) but higher than those of the rMOMP (39.83%) and PBS (35.85%) groups (Fig. 5 A, D, G, and J).Pr olifer ating CD4 + Tcells (M1) were also similar and higher in numbers between the SC (40.37%) and IM-p (39.69%) than the rMOMP (31.69%) and PBS (25.87%) mice (Fig. 5 B, E, H, and K).An essential, memory (CD44 high CD62L high ) T-cell phenotype was induced by immunization of mice in both the SC (5.68%) and IM-p (5.36%), compared to the rMOMP (3.24%) and PBS (2.25%) groups (Fig. 5 C, F, I, and L).Ho w e v er, the CD4 + effector (CD44 high CD62L low ) phenotype was higher for the SC (16.21%) compared to IM-p (11.06%), rMOMP (10.95%) and PBS (5.41%) groups (Fig. 5 C, F, I, and L).
Similarl y, activ ated CD4 + T-cells increased more in the SC and IM-p mice after a genital challenge (Fig. 5 M, P, S, and V), follo w ed by the rMOMP and PBS mice.CD4 + T-cell numbers increased in the sequential order of magnitude SC > IM-p > rMOMP > PBS (51.63%, 48.33%.41.78%, and 36.72%),along with heightened pr olifer ation (r espectiv el y, 48.07%, 44.80%, 37.24%, and 30.70%) (Fig. 5 N, Q, T,  and W).The impact of a Cm challenge was also evident with the induction of more effector CD4 + T-cells in mice being higher in SC (38.61%), follo w ed b y IM-p (35.28%), rMOMP (31.79%), and PBS (25.56%) groups (Fig. 5 O, R, U, and X).Conv ersel y, a r eduction of the CD4 + T-cells memory phenotype was seen after challenge in SC (3.04%), IM-p (2.49%), rMOMP (1.76%), and PBS (1.99%) mice due to the higher numbers of effector cells (Fig. 5 O, R, U, and X).Together these findings suggest that both prime-boost strategies elicited cell-mediated immune effectors that correlated with their protected status.Ho w e v er, the SC homologous prime-boost induced the highest cellular immune effectors that possibly enhanced the protection of mice against a genital Chlamydia challenge.

Nano v accine-induced antigen-specific serum and mucosal antibodies in mice
Next, we e v aluated the humor al pr otectiv e imm unity induced in mice by measuring antigen-specific total IgG and Th1 (IgG2a and IgG2b) and Th2 (IgG1) systemic and mucosal antibody isotypes before and after a chlamydial genital challenge.Sera obtained fr om imm unized (pr e), and imm unized-c hallenged (post) mice were pooled per group to quantify rMOMP-specific antibody isotype endpoint titers by ELISA.Our results show that SC or IMp mice produced elevated (2-fold or more) IgG antibodies (pre or post) compared to the rMOMP mice (pre or post) (Fig. 6 A and B; Table 1 ).Higher Th1 (IgG2a and IgG2b) and Th2 (IgG1) IgG antibody isotypes wer e pr oduced in the SC than the IM-p mice after immunization with both exhibiting a mixed T h1/T h2 antibody profile .T he rMOMP-immunized mice (pre) also produced predominant Th1 than Th2 antibodies (Fig. 6 C, E, and G; Table 1 ).After a genital challenge (post), all antibody isotypes receded in the SC, but the IM-p mice had an enhanced Th1 with reduced Th2 antibodies.Only IgG2b was increased in rMOMP mice after challenge while other isotypes remained unchanged (Fig. 6 D, F, and H; Table 1 ).Except for a weak IgG2b production after a genital challenge, the PBS control mice (pre) did not produce antigen-specific antibodies (Fig. 6 A-H; Table 1 ).
Further, we e v aluated rMOMP-specific m ucosal antibodies by collecting mucosal washes from immunized (pre) and imm unized-c hallenged (post) mice, as described above for sera.We observed (Fig. 7 A and B; Table 2 ) that SC and IM-p immunizations (pre) induced 2-4-fold higher IgG antibody titers that increased by 128-fold after challenge in SC (post) relative to the rMOMP mice.Collectiv el y, all gr oups of imm unized mice (pr e) pr oduced low m ucosal IgG isotypes (Fig. 7 C, E, and G).The data shows an interesting pattern for mucosal responses in SC (post) with marked increase of Th1 (IgG2a; 2-fold and IgG2b; 16-fold), Th2 (IgG1; 128-fold), and IgA (2-fold) antibodies, whic h wer e not seen in other groups (Fig. 7 D, F, and H; Table 2 ).PBS mice (pre) did not produce antigen-specific mucosal antibodies (Fig. 7 A-J; Table 2 ).
Ov er all, our r esults demonstr ate that the PLGA deliv ery system enhanced the production of Th1 and Th2 antibod- ies against rMOMP at both systemic and mucosal sites in mice.Mor e importantl y, the SC homologous prime-boost str ategy was more effective in eliciting robust humoral protective immunity.
Evaluation of the T h1/T h2 ratios after immunization in the SC and IM-p mice r e v ealed that their IgG2a/IgG1 ratios (pre) were similar and suggestive of a Th2-type r esponse, whic h ske wed to w ar d a Th1-type response after challenge (post) for the IM-p and not SC mice (Fig. 8 A).The IgG2b/IgG1 ratios (pre) were similarly of the Th2-type in immunized (pre) mice, which skewed tow ar d the Th1-type after challenge (post) in the SC and IM-p mice (Fig. 8 B).On the contr ary, m ucosal T h1/T h2 antibody ratios after imm unization (pr e) wer e indicativ e of a Th1-type that were dominated after challenge (post) by Th2-type response (Fig. 8 C  and D).IgG2a pr oduction (pr e) did not r eac h an endpoint titer to calculate the IgG2a/IgG1 ratio for the IM-p mice (Fig. 8 C; Table 2 ).The rMOMP immunization induced lo w er Th1 responses and were not changed after a Cm challenge, except IgG2b (serum) and IgG2a (wash).PBS and rMOMP were not included in the gr a phs for clarity (Fig. 8 ).Ov er all, these r esults show that the SC and IM-p prime-boost immunizations induces T h1 response , whereas genital challenge because of bacteria skews it to a Th2 response (mucosal).

Nano v accine-induced antigen-specific avidity serum antibodies in mice
T he a vidity or functional affinity of antigen-specific antibodies induced by an effectiv e v accine r enders specificity for the inactivation of the pathogen.The boosting of antigen-specific antibodies by PLGA-rMOMP in immunized mice, especially SC, led us to measure the avidity of serum IgG isotypes as a correlate of the humor al pr otectiv e imm unity.We used ur ea at a molar concentr ation of 8 M as a c haotr opic a gent to r elease the low-affinity antibodies from antigen-antibody complexes.Our results show an increase of IgG2a avidity after immunization (pre) being higher in SC and then IM-p, and rMOMP (Fig. 9 A), with further increases after a genital c hallenge (post), notabl y in SC mice (Fig. 9 B).Both SC and IM-p mice (pre) had similar and higher IgG2b avidity as compared to rMOMP (Fig. 9 C), which slightly decreased in the SC, r emained unc hanged in IM-p, but incr eased in the rMOMP mice (post) after challenge (Fig. 9 D).IgG1 avidity in the SC mice (pre) was high in comparison to the IM-p or rMOMP mice (Fig. 9 E), which then dr asticall y r educed in the SC, but slightl y incr eased in the IM-p and rMOMP mice following challenge (post) (Fig. 9 F).These results show that immunization with PLGA-rMOMP elicited high a vidity antibodies .T he high a vidity T h1 antibodies produced by the SC homologous prime-boost may correlate as a measure of their biological functions in the humoral protective immunity of mice against a genital challenge.

Nano v accine-induced serum antibodies neutr aliza tion of EB
Neutralizing antibodies bind to the surface of EBs and pr e v ent the infectivity of the cells .T her efor e, assessment of neutr alizing antibodies fr om ser a of imm unized (pr e) and imm unized-infected (post) mice was performed in vitro .EBs were preincubated with sera and then added to the confluent layer of McCoy cells, following 30 h incubation to allow the de v elopment of inclusions .T he results (Fig. 10 A) show that the SC or IM-p imm unization (pr e) induced antibodies that significantly ( P < .0001)neutralized Cm EB, when compared to the PBS group.Moreover, sera from both SC and IM-p mice (post) were significantly ( P < .0001)more effective in neutralizing Cm EB in comparison to the non-immunized PBS (post) gr oup.Ev en though, the ser a fr om rMOMP-imm unized (mice pr e) significantl y ( P < .1)neutr alized Cm EB, ther e was no significant increase after challenge (post).In addition, the rMOMP sera sho w ed significantly ( P < .01 and P < .0001)less neutralization of EB compared to those of the SC (pre and post) and ( P < .1)IM-p groups (post).
We also analyzed the immunized (pre) and immunizedchallenged (post) % sera neutralization of EB (Fig. 10 B) in each group of mice .T he analysis shows that SC and IM-p (pre) sera neutr alized c hlamydial EB by 55% and 42%, r espectiv el y, compar ed to the PBS gr oup.Mor eov er, the SC and IM-p imm unized-c hallenged (post) sera enhanced the neutralization of EB by 77% and 64%, r espectiv el y. rMOMP-imm unized mice (pre) sho w ed 26% neutralization with an increase to 34% after challenge (post) compared to the PBS gr oup.Ov er all, the r esults show that the SC and IMp immunization routes induce neutralizing antibodies.Ho w ever, SC-r oute induced mor e than 20% additional neutralizing antibodies compared to the IM-p route.

Discussion
Ov erwhelming r esearc h efforts to de v elop a subunit v accine against Ct has encountered significant hurdles, including delivery platforms , adjuvants , and administr ation r outes (Ralli-Jain et al. 2010, Fiorino et al. 2013, Dixit et al. 2014, Stary et al. 2015 ), to elicit efficacious pr otectiv e imm unity.Routes of v accine administr ation ar e critical for inducing efficient cell-mediated and humor al imm une r esponses to pr otect a gainst a pathogen.Importantl y, pr otection a gainst Ct is dependent on r obust CD4 + T-cells and antibody effector responses for bacterial clearance (Farris et al. 2010 ).An effectiv e imm unization str ategy may r equir e primeboost deliv ery r outes to influence the persistence of antigens and enhance T-and B-cell responses (Zacharias et al. 2018 ).Studies have shown that the SC route administration is considered appr opriate for deliv ering antigens due to efficient dr aining to l ymphoid organs to activate immune responses (Zhao et al. 2023 ).Notabl y, the SC r oute is gaining attention for the extended delivery of drugs and subunit vaccines using biodegradable nanoparticles (Taha et al. 2012, Van de Ven et al. 2012, Fairley et al. 2013, Zakeri-Milani et al. 2013, Dixit et al. 2014, Singh et al. 2015, Verma et al. 2018, Sahu et al. 2020 ).Likewise, subunit vaccines are also paving the w ay to w ar d an IM immunization route that is more widespread for commercial vaccines (Herzog 2014, Ols et al. 2020 ) due to ease of administration and acceptability .Recently , Ols et al. ( 2020 ) demonstrated in nonhuman primates that the SC and IM imm unization r outes induced early differences in HIV-1 glycoprotein antigen trafficking with SC delivering to primary and IM to secondary lymph nodes (LNs); ho w ever, with similar induction of antigen-specific cellular and humoral responses.
A variety of immunization routes are being tested for a Ct vaccine (Badamchi-Zadeh et al. 2016, Wern et al. 2017 ), albeit some may undeniably and humanly impose challenges .T herefore , vaccines pr efer entiall y employing the most common imm unization r outes a ppr oac h ar e al ways desir able.We pr e viousl y r eported that SC immunization of our chlamydial PLGA-rMOMP nanovaccine induced robust adaptive immune responses in mice along with memory and effector formation (Sahu et al. 2020 ), but did not afford complete protection of mice against a Cm genital challenge (Sahu et al. 2021 ).Giv en that v accine deliv ery r outes can a ppr eciably impact the outcome of efficacious pr otectiv e imm une r esponses, here in the present study, we explored two prime-boost imm unization str ategies to compar e the c hlamydial nanov accineinduced immunogenicity and protective efficacy in mice against a chlamydial genital challenge.
We have demonstrated here that SC (homologous) and IMp (heterologous) prime-boost immunization strategies effectively pr otected mice a gainst genital Chlam ydia by enhancing an early bacterial clearance and reduced bacterial burdens in contrast to the rMOMP and control mice.Ho w ever, the IM-p mice had higher bacterial burdens, pr obabl y due to a lac k of toler ance a gainst establishing a Cm infection.Conv ersel y, the SC mice pr e v ented the establishment of infection by the reduced bacterial burden and earl y clear ance of infection with total of 88% IFU r eduction, suggesting enhanced protection afforded by the homologous immunization.Ralli-Jain et al.  the poliovirus v accine (Holmgr en and Czerkinsky 2005 ).Se v er al Chlam ydia v accines in pr eclinical de v elopment similarl y ar e being administered via the mucosal route (Pal et al. 1996, Manam et al. 2013 ).Herein, our findings reveal a paradigm shift from a mucosal pathogen mandating a m ucosal r oute for vaccine administration and efficacy, which could be attributed to the PLGA delivery system's versatility for various immunization routes to elicit protection against genital Chlamydia .Earlier research indicated mandatory r equir ements for T-cells with a minimal role for antibodies in pr otectiv e imm unity and clear ance of Chlam ydia (Su andCaldwell 1995 , Li et al. 2008 ).Now compelling evidence reveals that pr otection a gainst Chlam ydia r equir es cell-mediated and humor al immune effectors for bacterial clearance (Farris et al. 2010 ).
The role of antigen-specific CD4 + T-cells producing Th1 cytokines, particularl y IFN-γ is well-r ecognized in the clear ance of Chlamydia (Lin et al. 2019, Tifrea et al. 2020 ).In the current study, we observed that T-cells from SC and IM-p mice secreted higher le v els of rMOMP-specific IFN-γ after immunization and challenge compared to the rMOMP and control mice.IFN-γ production accompanied by IL-2 is a clear indicator of T-cell pr olifer ation and is congruent with our pr e vious r eports of PLGA-rMOMP (Sahu et al. 2021 ) or PLA-PEG-M278 (a peptide of MOMP) (Verma et al. 2018 ) immunization of mice via the SC-route that exhibited a predominant Th1 immune response .T he presence of IL-2 along with IFN-γ is a dynamic relationship as IL-2 dir ectl y acts to stimulate T-cells to produce IFN-γ (Kasahara et al. 1983 ), a requisite cytokine for pr otection a gainst Chlam ydia (Helble et al. 2020 ).Ther efor e, the enhanced IL-2 and IFN-γ production in the current study underscores the described relationship in the above study.We also observ ed an upr egulated pr oduction of the imm unor egulatory IL-17 cytokine in the SC, IM-p, and rMOMP immunized mice (Pre) facilitated by the PLGA-rMOMP nano vaccine , and in all groups of mice after Cm genital c hallenge, whic h is similar to our pr e vious findings (Sahu et al. 2021 ).IL-17 is a common cytokine produced by T h17 T-cells , howe v er its r ole in Chlam ydia infection is still debatable between protection and pathology.Andr e w et al. ( 2013) study demonstrated that IL-17 KO mice that were infected with Chlamydia and then immunized intranasally with MOMP, cholera toxin and CpG adjuvant were unable to clear the infection and exhibited less pathology, IFN-γ production, and T-cell pr olifer ation.Upregulation of IL-17 producing T-cells also has been linked with IL-6 producing T-cells as reported by Moore-Connors et al. ( 2013 ) and Zhou et al. ( 2013 ) in Chlamydia .The protection afforded by SC and IM-p groups of mice may be correlated with IL-6 regulation of IL-17 coupled with increased T-cell producing IFN-γ , which are induced by IL-2 pr olifer ation.It can be said that IL-17 plays an important role in protection but may not be directly associated with pathology.
We also observed that immunized and protected mice produced IL-4, IL-9, and IL-13, all T h2 cytokines , albeit at lo w er le v els compared to the predominant levels of the Th1 cytokines, IFN-γ and IL-2, which underscores our previous observation for immunization via SC or IN routes (Sahu et al. 2021 ).In addition, elev ated le v els of G-CSF wer e seen in SC mice (pre) follo w ed b y IM-p, whic h wer e further incr eased after Cm c hallenge, as well as in other groups of mice.In general, G-CSF functions as survival, prolifer ation and differ entiation of neutr ophils and inducing leuk ocytes cell production from bone-marrow (Link 2022 ) but its role in Chlamydia is yet to be defined.Of note, SC and IM-p mice exhibited rMOMP-specific CD4 + T-cell activ ation, pr olifer ation, and differentiation into memory (CD44 high CD62L high ) phenotypes but with a higher effector (CD44 high CD62L low ) phenotype in SC, which is consistent with our pr e vious r eport (Verma et al. 2018, Sahu et al. 2021 ) and others (Li et al. 2008, Helble et al. 2020 ).Inter estingl y, CCL-2 production (Fig. 3 C) in SC or IM-p mice (pre) is additional evidence of inducing recruitment of memory T-cells, especially IFNγ producing T-cells.It was reported that Chlamydia induce CCL-2 production (Belay et al. 2002, Schrader et al. 2007 ), ho w ever, CCL-2 production by SC and IM-p immunization is an interesting finding in the current study.The differences in cellular responses between SC and IM-p mice may infer differences in antigen processing, as demonstrated in nonhuman primates with SC targeting primary LNs and IM secondary LNs (Ols et al. 2020 ).Our recent publication r e v ealed that PLGA-rMOMP incr eased MHC class II antigen presentation and targeted primary LNs in SC-immunized mice (Sahu et al. 2020 ).Collectiv el y, we could speculate that efficient processing with higher cell-mediated immune effectors could, in part, explain the better protection of SC than the IM-p mice against genital Chlamydia .
That antibodies are essential for the clearance of Chlamydia was elegantl y demonstr ated by Farris et al. ( 2010) in B-cell deficient mice lac king v accine-induced pr otection and by P al et al. ( 2005) linking Chlamydia -specific Th1 (IgG2a and IgG2b) antibodies to pr otection.Her ein, pr otection of the SC and IM-p mice involved the engagement of Th1 (IgG2a and IgG2b) and Th2 (IgG1) antibodies, given their high antibody titers, especially in SC mice.Inter estingl y, Th2-type antibody r esponses dominated after imm unization with a bias to w ar d Th1-type after challenge, facilitated by the bacterial infection.After immunization, the dominant Th2 antibodies may have ensued from the self-adjuvanticity of PLGA since mice immunized with rMOMP induced mainly Th1 antibodies.Even though, we observed a reduction in rMOMP-specific systemic antibodies titer after Cm challenge in SC mice, this contrasted with the IM-p mice.Ne v ertheless, SC and IM-p mice induced mixed T h1/T h2 antibodies , but only T h1 antibodies exhibited high avidity, especially in SC mice .T his finding, further correlates with Th1 isotypes pr e v enting establishment of early infection (Hawkins et al. 2002, Ralli-Jain et al. 2010 ) and supposedly the differences in the protection levels between the IM-p and SC mice in clearing genital Chlamydia .
In this study, higher rMOMP-specific IgA occurred in the IM-p than SC mice, a pattern we pr e viousl y observ ed in PLGA-rMOMP SC-immunized mice (Sahu et al. 2021 ).Results from studies indicate antigen-specific IgA can only provide a partial reduction of c hlamydial infections (Armita ge et al. 2014, Erneholm et al. 2019 ).Armitage et al. ( 2014 ) revealed that rMOMP-specific IgA antibodies r educed c hlamydial infection by 44% in the absence of CD4 + T-cells .T here is documentation of IgA and IgG producing plasma cells in the genital tract of pigs following an IM vaccination with UV-inactivated bacteria/CAF01 and a Chlamydia intravaginal challenge (Erneholm et al. 2019 ).A vaccine developed by Jiang et al. ( 2017 ) comprised of multiepitopes peptides of MOMP with Hepatitis B virus core antigen (HBcAg) enhanced immunogenicity with increased IFN-γ , IgG, and IgA effectors that improved efficacy and clearance of genital infection earlier than the contr ols.Pr esumabl y, the pr otectiv e imm unity a gainst Chlam ydia infection r equir es a synergistic effort facilitated by cellular and humor al imm une responses, as shown here, since specific antibodies only partially reduce the Chlamydia mucosal burden (Darville et al. 2019 ).
Onset of neutralizing antibodies to clear the pathogen directly correlates with memory B-cells (Young and Brink 2021 ).As a result, these memory cells are responsible for inducing humoral pr otectiv e r esponses a gainst pathogen and ar e important for an efficacious vaccine.Serum antibody-mediated Chlamydia neutralization in vitro is an important tool to predict the effectiveness of a Chlamydia vaccine (de la Maza et al. 2017 ).There are mul-tiple reports indicating that promising Chlamydia vaccine candidates induce neutralizing antibodies (Olsen et al. 2021, Tifrea et al. 2021, Zuo et al. 2021 ).Our results show that the levels of EB neutralization enhanced after the Cm intravaginal challenge, which are similar to our previous studies (Verma et al. 2018, Sahu et al. 2021 ).Chlamydial MOMP is the most proposed vaccine candidate for Chlamydia (Sahu et al. 2021, Huynh et al. 2022, Pal et al. 2023 ) and have shown that MOMP-specific antibodies possess neutralization functionality (Collar et al. 2022 ).In this study, we demonstrated that the nanovaccine homologous (SC) route is superior ov er the heter ologous (IM-p) priming imm unization r oute a gainst a Cm genital challenge.

Conclusions
In conclusion, the homologous SC prime-boost immunization of mice with PLGA-rMOMP induced higher cell-mediated and humor al imm une r esponses and conferr ed better pr otection a gainst a Cm genital challenge compared to the heterologous IM-p.This study is the first to report the comparison of IM-p versus SC primeboost immunization routes for immunogenicity and protective efficacy in Chlam ydia v accine de v elopment str ategy.With further optimization, perhaps including an adjuvant, PLGA-rMOMP holds promise as a nanovaccine candidate that can confer even higher pr otection a gainst genital c hlamydial infections.
Wang X , Singh AK, Zhang X et

Figure 1 .
Figure 1.Schematic of immunization and challenge .(A) F emale BALB/c mice (six per group) were each given PLGA-rMOMP (50 μg) on day 0 for priming (p) via the IM-p (heterologous) or SC (homologous) routes.IM-p and SC mice each received two boosters (b) immunization of PLGA-rMOMP (50 μg) via the SC route on days 14 and 28 and then sacrificed on day 42 (six per group) for immunogenicity studies.(B) For efficacy studies, immunized mice (six per group) were each challenged via the intravaginal route with live Cm IFU (1 × 10 5 ) on day 49, follo w ed b y a collection of cervico-v a ginal swabs at 3-day intervals up to 3-weeks to quantify Cm IFU followed by sacrifice on day 70.Some mice r eceiv ed rMOMP (50 μg) or PBS via the SC route to serve as contr ols.After eac h sacrifice (days 42 and 70), spleens (for T-cells), blood (for serum), and mucosal w ashes w er e collected to e v aluate cellular and humor al imm une r esponses befor e and after a c hallenge.(Illustr ation cr eated in Bior ender.com).

Figure 2 .
Figure 2. SC rather than the IM-p prime-boost immunization provides better protection against a genital chlamydial challenge.Mice were each given PLGA-rMOMP (50 μg) as priming via the IM-p (heterologous) or SC (homologous) routes.IM-p and SC mice each received two booster immunization of PLGA-rMOMP (50 μg) via the SC route at 2-week intervals and then challenged via the intravaginal route with live Cm IFU (1 × 10 5 ).Cervico-vaginal sw abs w er e collected at 3-day interv als up to 3-weeks and pr opa gated in McCoy fibr oblasts to quantify r ecov er ed Cm IFU fr om s wabs .(A) Each floating bar r epr esents the minim um and maxim um r ange for the IFU counts (IFU/ml) fr om individual s wabs , and the horizontal mid dle dotted line re presents the mean of IFU/ml for each group of mice after challenge.(B) Graph insert represents the average of total IFU/ml (mean ± SE) calculated for each group between days 3 and 18, and presented as a % reduction of IFU compared to the PBS control.Immunofluorescence microscopic visualization of Cm IFU (green) cytoplasm in fibroblasts (red).Fibroblasts were exposed to swabs collected from mice on day 12 after the challenge.(C) PBS, (D) rMOMP, (E) IM-p, and (F) SC groups.Statistical analyses were performed using tw o-w ay ANOVA follo w ed b y Tuk e y's P ost-test (A) and a v er a ge IFU comparison was performed using one-way ANOVA (B).Significant differences in IFU counts were considered at * P < .05,* * P < .01,and * * * * P < .0001.No exclusions wer e a pplied for IFU counts.

Figure 6 .
Figure 6.Production of systemic rMOMP-specific antibodies after immunization (pre) and challenge (post).Groups of mice were immunized and challenged, as described in the legend of Fig. 1 .Sera collected from groups of immunized (pre), and immunized-challenged (post) mice were pooled per group and used to quantify rMOMP-specific antibody isotypes by ELISA.Immunized mice (pre); (A) IgG, (C) IgG2a, (E) IgG2b, and (G) IgG1, and imm unized-c hallenged mice (post); (B) IgG, (D) IgG2a, (F) IgG2b, and (H) IgG1.Sera were diluted at a 2-fold serial dilution to determine the endpoint antibody isotype titers.Each data point represents the mean ± SD of triplicate samples.

Figure 8 .
Figure 8. Serum and mucosal wash T h1/T h2 antibody ratios after immunization (pre) and challenge (post).Groups of mice were immunized and challenged, as described in Fig. 1 legend.Sera collected from groups of immunized (pre), and immunized-challenged (post) mice were pooled per group and used to quantify rMOMP-specific antibody isotypes by ELISA.Serum and mucosal antibodies endpoint titers were used for calculating the T h1/T h2 ratios; (A) and (B) serum, (C) and (D) mucosal wash.
( 2010 ) investigated systemic (IM and SC) and mucosal (sublingual and colonic) immunization routes, alone and in combinations using c hlamydial rMOMP, a gainst a r espir atory c hlamydial c hallenge .T heir r esults r e v ealed that the combined mucosal and systemic routes were most effective, especially a simultaneous combination of the sublingual, IM, and SC routes (Ralli-Jain et al. 2010 ).Similarly, Carmichael et al. ( 2011 ) reported that a combined systemic and intr av a ginal m ucosal r oute enhanced protection against a Chlamydia genital challenge.Howe v er, the m ucosal r outes r equir e a high antigen dose for immunization due to r a pid clear ance (de la Maza et al. 2017 ).Ideall y, m ucosal routes are more favorable for mucosal pathogens to induce local pr otectiv e imm une effectors.Indeed some pr eclinical v accines a gainst m ucosal pathogens do employ m ucosal r outes suc h as intranasal for Yersinia pestis (Wang et al. 2020 ), influenza virus (Quan Le et al. 2020 ), and Leishmania (infantum) chagasi (Leal et al. 2015 ).Or al imm unization is also acceptable for some vaccines , i.e .